- Rene - PCR clone library
- Ryan - cultures
- Cas-tone - cultures from streaks; Addgene plasmids, 5mL each, overnight; 7 total (4 amp, 3 kan)
Rene - PCR clone library
- Use the CloneJET PCR Cloning Kit - MAN0012707 CloneJET PCR Cloning (manual)
- pJET1.2/blunt vector is 5'-phosphorylated and accepts blunt products (Phusion PCR)
- All common laboratory E. coli strains can be directly transformed with the ligation product
- Re-circularized vector expresses toxic enzyme, no blue/white screening needed
- Optimal insert : vector (0.05 pmol ends) ratio is 3:1; Calculation: length of insert DNA x 0.05 = ng insert to use
- Ligations - CloneJET PCR Cloning Kit
- Luc14 + CRISPR g034; size = 630
- Gal4EED/luc + CRISPR g034 = 630
| 2x reaction buffer || 10.0
| PCR product (31.5 ng) || 2.0
| pJET1.2/blunt vector || 1.0
| T4 ligase || 1.0
| dH2O || 6.0
| || 20.0 μL
- Incubate the ligation mixture at room temperature (22°C) for 5 min.
- Transform DH5α-turbo with 10 μL ligation reaction. Follow quick protocol.
- Plate on 100 μg/mL amp.
- Worked, but not very many colonies
- Redo ligation with 2x as much insert
- Follow long transformation protocol (heat-shock & recovery in SOC)